Retinoic acid inducible gene-I (RIG-I) plays important roles during innate immune responses to viral infections and as a transducer of cytokine signaling. The mechanisms of RIG-I up-regulation after cytokine stimulation are incompletely characterized. It was previously reported that IFN–γ induces the expression of RIG-I in endothelial cells. In this study, we characterized the mechanism of type I IFN-mediated up-regulation of RIG-I in HeLa cells and found that, in addition to type I IFN, TNF-α, a cytokine that regulates innate immune responses, induced expression of RIG-I. To investigate whether TNF-α-and type I IFN-mediated up-regulations of RIG-I were causally related, we studied the kinetics of these responses. Our results were consistent with a model in which TNF-α functioned upstream of type I IFNs. The ability of TNF-α to up-regulate RIG-I required protein synthesis, expression of functional type I IFNRs, and STAT1 signaling. We also found that IFN-ε was the only IFN isoform expressed constitutively in HeLa cells and that its expression was up-regulated in response to stimulation with TNF-α. The mechanism of up-regulation involved stabilization of IFN-ε mRNA in the absence of transcriptional activation. Silencing the expression of IFN-ε attenuated STAT1 expression and phosphorylation and inhibited RIG-I expression, providing additional support for the participation of IFN-ε upstream of STAT1. Our findings support a sequential mechanism whereby TNF-α leads to stabilization of IFN-ε mRNA, increased IFN-ε synthesis, engagement of type I IFNRs, increased STAT1 expression and phosphorylation, and up-regulation of RIG-I expression. These findings have implications for our understanding of the immune responses that follow cytokine stimulation.